Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented. / Master of Science

Bovine viral diarrhea virus (BVDV) is a pestivirus that suppresses the innate and adaptive host immune responses. Each of the two classified genotypes (BVDV1 and BVDV2) has two distinct biotypes – cytopathic (cp) and non-cytopathic (ncp) – and evidence has suggested that cytopathic strains may disrupt host interferon (IFN) synthesis and IFN-mediated responses. However, inconsistent results examining ncpBVDV strains have generated controversy regarding whether they also exhibit this capability. The purpose for this study was to determine the occurrence and functionality of IFN-induced responses within the serum cattle infected with ncpBVDV2-1373. Specifically, this involved analysing the changes in both the serum levels of IFN-α and IFN-γ and the expression of genes that are classically regulated by these cytokines. Serum analysis showed that the infected cattle induced both serum IFN-α and IFN-γ during BVDV infection while PBMC analysis showed increased expression of genes that classically respond to IFN-α – Mx-1, OAS-1, and STAT-1 – and IFN-γ – SOCS-1 and SOCS-3. These findings are supported by temporal kinome analysis, which verified activation of the JAK-STAT signalling network within the PBMCs of the virus-infected animals. In addition to establishing evidence for its synthesis, results from this challenge identified IFN-γ as a possible indicator of animal mortality as analysis of its change within the non-surviving, infected animals was statistically greater than the levels of the surviving, infected animals. Collectively, these results demonstrate 1373-mediated induction of, and host cell response to, both IFN-α and IFN–γ, and the potential for IFN-γ to be a predictive marker for mortality during BVDV infection.

Drosophila melanogaster has been extensively used as a model organism to study diverse facets of biology, including host-pathogen interactions and the basic biology of its pathogens. I have used the fruit fly as a model to study elementary aspects of Nora virus biology, such as the role of the different proteins encoded by the virus genome. Nora virus, an enteric virus transmitted via the feca-oral route, does not cause any obvious pathology in the fly, although the infection is persistent. Nora virus genome consists of a positive strand RNA that is translated in four open reading frames (ORF).  Since sequence homology studies did not yield much information about the different Nora virus proteins, I have used the cDNA clone of the virus to construct mutants to identify the specific function of each protein. My results have shown that, 1) The protein(s) encoded by ORF 1 are crucial for the replication of the virus genome. 2) The C-terminus of the ORF 1-encoded protein (VP1), is an inhibitor to the RNAi pathway. 3) The transmembrane domain in the N-terminus of the ORF2-encoded protein (VP2) is important for the formation of Nora virus virions. 4) The ORF 3-encoded protein (VP3) forms α-helical trimers and this protein is essential for the stability of Nora virus capsid.                                                     I have also performed RNA sequencing to investigate the transcriptional response of D. melanogaster in response to Nora virus infection and my results indicate that,                        5) The upregulation of genes related to cellular stress and protein synthesis and the downregulation of basal digestive machinery, together with the induction of upd3, implies major gut epithelium damage and subsequent regeneration.

Pathogenic Listeria are the causative agents of listeriosis, a severe food-borne infection. They are able to invade various non-phagocytic cell types including epithelial cells. The life cycle of these intracellular parasites involves penetrating into host cells, rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly spreading cell to cell. Each step of the listerial intracellular infection involves activation of virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated virulence gene activation occurs within host cells by mechanisms that remain unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen interaction. Methods for assessing PrfA-dependent gene expression were first developed and standardized, including a highly sensitive and accurate quantitative reverse-transcription real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with virulence using cell culture-based assays. These techniques were applied in an investigation into the structure-function of PrfA. We studied the role of a solvent-accessible pocket identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding (CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene activation. Site-directed PrfA mutants were constructed. Our data support the notion that PrfA activity is allosterically regulated and are consistent with a role for the pocket as putative binding site for the PrfA-activating allosteric effector. The characterization of spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*- suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated repression was investigated and found to explain the anomalous phenotype of strain NCTC 7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by cellobiose but not glucose. We also carried out experiments to address the intriguing activation of PrfAdependent virulence genes upon addition of an adsorbent to the culture medium, the socalled "charcoal effect". Using a chemically defined culture medium and resin, Amberlite™ XAD-4, we provided evidence that the virulence gene activation may involve the sequestration of a medium component rather than a bacteria-derived autorepressor, as initially thought. We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We showed that σB has no major effect on host cell invasion, and that L. monocytogenes invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in stress conditions. Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and developed normalized methods to accurately determine and quantify this cellular response in infected cell monolayers.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

No description available.

Catfish, the "king" of the U.S. aquaculture, is threatened by a severe, systemic bacterial disease known as enteric septicemia of catfish (ESC). This disease causes high mortality and massive economic losses in cultured channel catfish (Ictalurus punctatus) in the United States. E. ictaluri penetrates catfish intestinal epithelia quickly and establishes a systemic infection rapidly. However, our knowledge on catfish intestine and E. ictaluri interaction is very limited. In Particular, catfish intestinal immune responses and virulence genes needed by E. ictaluri to evade host defenses are not well understood. Hence, our long-term goal is to identify the molecular mechanisms of E. ictaluri-host interactions. The overall objectives of this study were to understand catfish immune responses to E. ictaluri infection and determine essential genes of E. ictaluri during the intestinal invasion. To accomplish the overall objectives of this research, intestinal ligated loops were constructed surgically in live catfish and loops were injected with wild-type E. ictaluri and two live attenuated E. ictaluri vaccine strains developed recently by our research group. We first determined catfish intestinal immune responses against E. ictaluri wild-type and live attenuated vaccine strains. Then, we analyzed the global gene expression patterns of wild-type E. ictaluri and vaccine strains during catfish intestinal invasion using high throughput RNA-Seq technology. Results showed a moderate level of neutrophil and B cell infiltration correlated with significantly lower expression of TNF-α, CD4-1, and CD8-α in the vaccine injected intestinal tissue compared to that of wild-type injected intestinal tissue. Further, RNA-Seq data analysis showed the prominent expression of genes related to bacterial secretion systems, ATP production processes, and multidrug resistance (MDR) efflux pumps in wild-type E. ictaluri. In contrast, the prominently expressed genes in vaccine strains were related to the phosphotransferase system and sugar metabolism processes. All these data suggest that our live attenuated vaccines are capable of triggering effective immune responses in catfish without causing damage to the host.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.